The present invention concerns a cloned human neurokinin-3 receptor (hereinafter identified as human NK3R).
Neurokinin B (NKB) is a naturally occuring peptide belonging to the neurokinin family of peptides, which also includes substance P (SP) and substance K (SK). NKB binds preferentially to the neurokinin-3 receptor (NK3R), although it also recognizes the other two receptor subtypes (NK1 and NK2) with lower affinity. As is well known in the art, neurokinin B and other tachykinins have been implicated in the pathophysiology of numerous diseases. Neurokinin peptides are reportedly involved in nociception and neurogenic inflammation. The physiological function of NK3R has been implicated in the regulation of enkephalin release, while the NK1 and NK2 receptor subtypes are involved in synaptic transmission (Laneuville et al., Life Sci., 42:1295-1305 (1988)). Since the NKB genomic structure and subcellular distribution are different from those of SP and SK, the physiological function and regulatory mechanism of NKB may be different from SP and SK.
More specifically, neurokinin B is a pharmacologically-active neuropeptide that is produced in mammals and possesses a characteristic amino acid sequence that is illustrated below:
Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2. PA1 (a) a mammalian expression vector, such as pcDNAI/Neo, and PA1 (b) a base sequence encoding human neurokinin-3 receptor protein. PA1 (a) a mammalian expression vector, such as pcDNAI/Neo, and PA1 (b) a base sequence encoding human neurokinin-3 receptor protein. PA1 (a) a mammalian expression vector, such as pcNDAI/Neo and PA1 (b) a base sequence encoding human neurokinin-3 receptor protein. PA1 which plasmid comprises: PA1 which plasmid comprises: PA1 which plasmid comprises:
Several groups have reported the cloning of certain neurokinin receptors. T. M. Fong, et al., Mol. Pharmacol., 41:24-30 (1991) have reported cloned human neurokinin-l and neurokinin-1 short form receptor. J. Yokota, et al., J. Biol. Chem., 264:17649 (1989) have reported cloned rat neurokinin-1 receptor. N. P. Gerard, et al., J. Biol. Chem., 265:20455 (1990), have reported human neurokinin-2 receptor. Cloned rat and bovine neurokinin-2 receptor have likewise been reported. See respectively, Y. Sasi, and S. Nakanishi, Biochem Biophys. Res. Comm., 165:695 (1989), and Y. Masu, et al., Nature 329:836 (1987). Cloned rat neurokinin-3 receptor has been reported by R. Shigemoto, et al., J. Biol. Chem., 265:623 (1990). The above references, however, neither disclose nor suggest the present invention.
The instant invention also concerns an assay protocol which can be used to determine the activity in body fluids of substances that bind human NK3R; these include neurokinin B. The assay can also be used for identifying and evaluating substances that bind NK3R. Thus, the assay can be used to identify neurokinin B antagonists and evaluate their binding affinity. Another method for an assay includes that described by M .A. Cascieri, et al., J. Biol. Chem., 258:5158 (1983). See also, for example, R. M. Snider, et al., Science, 251:435 (1991) and S. McLean, et al., Science, 251:437 (1991). See also WIPO Patent Publications WO90/05525 and WO90/05729, published May 31, 1990. Methods to date have proven inferior, in part, for failure of the animal receptor (animal NK1R, NK2R or NK3R) activity to accurately reflect that of the human neurokinin-3 receptor. Furthermore, prior to this disclosure, human NK3R has not been available in a highly purified form or in substantial isolation from NK1R and/or NK2R. Use of such neurokinin receptor sources can not accurately depict the affinity of an agonist or an antagonist for a human NK3R.